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EFNA3 / Ephrin-A3 · IHC design guide
Design Immunohistochemistry for EFNA3
Plan chromogenic EFNA3 IHC in paraffin sections using breast glandular cells or cerebral cortex neurons as high-staining references (HPA tissue IHC). Compare the reported cytoplasmic staining (HPA tissue IHC) with EFNA3’s GPI-anchored membrane location (UniProt), and interpret results cautiously because HPA rates the tissue evidence uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EFNA3 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining; reliability uncertain (HPA tissue IHC) | |
| Regulation | No specific regulatory cue reported (UniProt) | |
| Isoform / epitope | 2 isoforms; extracellular chain 23–214, no cytoplasmic tail (UniProt) |
Section 1
Recommended EFNA3 IHC & IF Protocols
The catalog antibody protocol is accompanied by published EFNA3 chromogenic IHC protocols for bladder, lung and tumor sections (PMC10623084; PMC8513303; PMC13523511).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A07595-1) |
| Fixation | Image fixative and duration unreported (datasheet A07595-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-EFNA3, 1:50-1:200 (datasheet A07595-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EFNA3-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); adjust for the cited protocol when reproducing it.
Section 2
What Is the Expected EFNA3 Staining Pattern?
EFNA3 is a cell-surface, GPI-anchored ligand with no transmembrane segment (UniProt P52797 topology/function). In paraffin-section IHC, assess membrane-associated staining in the relevant cells, including breast glandular and cerebral-cortex neuronal cells reported as High by HPA (HPA: tissue IHC). Interpret that expectation cautiously: HPA describes cytoplasmic staining across several tissues and rates its tissue IHC reliability Uncertain because staining and RNA expression have low consistency and presumed off-target binding was observed (HPA: tissue IHC reliability).
| Membrane-associated staining in breast glandular or cerebral-cortex neuronal cells, with limited background. | This fits EFNA3's cell-surface localisation and the cell populations HPA scored High (UniProt P52797 topology; HPA: tissue IHC). It is a plausible positive pattern, not independent proof of antibody specificity: HPA rates the tissue IHC profile Uncertain (HPA: tissue IHC reliability). |
| Predominantly nuclear staining or strong, uniform cytoplasmic staining without discernible membrane enrichment. | Nuclear localisation conflicts with the UniProt cell-membrane annotation (UniProt P52797 subcellular location). Cytoplasmic staining needs more care: HPA reports it in several tissues, but flags presumed off-target binding and uncertain reliability. Compare the distribution with controls before assigning it to EFNA3 (HPA: tissue IHC profile/reliability). |
| Strong staining in a cell population HPA lists as Not detected, such as adipocytes or bone-marrow hematopoietic cells. | This conflicts with those HPA tissue IHC observations and raises possible cross-reactivity or endogenous chromogenic detection activity (HPA: tissue IHC; general IHC practice). HPA's Uncertain reliability means these populations are comparison points, not definitive EFNA3-negative controls (HPA: tissue IHC reliability). |
| Diffuse color across cells and surrounding tissue, obscuring cell boundaries. | The slide cannot support a compartment call. Background may arise from nonspecific antibody binding or the chromogenic detection workflow (general IHC practice). Review a matched control and the staining distribution before scoring EFNA3; HPA's Uncertain profile cannot resolve an ambiguous slide (HPA: tissue IHC reliability). |
| No signal in a breast section's glandular cells or a cerebral-cortex section's neuronal cells. | Both populations were scored High by HPA, so absence warrants a run-level check (HPA: tissue IHC). Confirm section quality, retrieval and detection with appropriate controls (general IHC practice). A blank result alone does not establish EFNA3 absence, especially given the Uncertain reference profile (HPA: tissue IHC reliability). |
💡Expected EFNA3 appearanceA plausible positive shows discernible membrane-associated color in breast glandular or cerebral-cortex neuronal cells scored High by HPA; isolated nuclear color or broad staining of HPA Not detected populations is suspect, and specificity remains unconfirmed (UniProt P52797 topology; HPA: tissue IHC/reliability).
| Compartment and topology | EFNA3 is a GPI-anchored cell-surface ligand without a transmembrane segment; use membrane association as the localisation benchmark (UniProt P52797 topology/function). |
| Tissue and cell selection | HPA scores breast glandular and cerebral-cortex neuronal cells High, while adipocytes are Not detected; use these as comparisons with the Uncertain reliability caveat (HPA: tissue IHC). |
| Antibody evidence | The listed antibody, CAB010494, has an Uncertain IHC assessment; its staining cannot by itself verify the expected pattern (HPA: antibody validation). |
| Processing and epitope location | The annotated mature chain spans residues 23–214 after signal-peptide and propeptide processing. Epitope position is unspecified, so predict no antibody-specific pattern from processing alone (UniProt P52797 processing). |
| Glycosylation and isoforms | UniProt records three glycosylation sites and two isoforms. Their effects on this antibody's paraffin-section staining are unreported; avoid assigning intensity differences to either (UniProt P52797 glycosylation/isoforms). |
| IF/ICC cross-check? | HPA's ICC-IF summary says Membrane, but gives no main location or cell-line images. It supports a localisation comparison, not an IF/ICC protocol or validation of this IHC result (HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Breast glandular cells stain, but color appears mainly nuclear. | The compartment conflicts with UniProt's membrane annotation; a source of nonspecific staining is possible (UniProt P52797 subcellular location; general IHC practice). | Review a matched detection control and the cell-boundary pattern. Report the nuclear result separately instead of scoring it as confirmed EFNA3 (general IHC practice; HPA: tissue IHC reliability Uncertain). |
| Cerebral-cortex neuronal cells show no detectable color. | HPA scored these cells High, but its tissue IHC assessment is Uncertain; a failed staining run or sample variation is possible (HPA: tissue IHC/reliability; general IHC practice). | Check section integrity and a successful run control, then review retrieval, antibody dilution and detection conditions as general IHC variables; do not infer an EFNA3-specific retrieval defect (general IHC practice). |
| Adipocytes or bone-marrow hematopoietic cells stain strongly. | HPA lists both as Not detected. Cross-reactivity or endogenous detection activity may explain discordant chromogenic color (HPA: tissue IHC; general IHC practice). | Compare a matched detection control and staining in HPA High cell populations. Treat discordance as a specificity question because HPA's own IHC reliability is Uncertain (general IHC practice; HPA: tissue IHC/reliability). |
| Diffuse background prevents assessment of membrane staining. | Nonspecific antibody binding or detection background can obscure localisation (general IHC practice). | Review blocking, antibody dilution, washes and detection controls; rescore only where cell boundaries can be resolved (general IHC practice; UniProt P52797 subcellular location). |
| Cytoplasmic color is widespread across otherwise plausible tissues. | HPA reports cytoplasmic expression in several tissues but also records low RNA–staining consistency and presumed off-target binding (HPA: tissue IHC profile/reliability). | Record the observed compartment and compare cell types and controls. Do not convert HPA's cytoplasmic description into proof of EFNA3-specific intracellular staining (HPA: tissue IHC reliability; general IHC practice). |
| IF/ICC membrane staining seems clearer than the IHC slide. | HPA labels its ICC-IF summary Membrane, yet supplies no main location or cell-line images; the IHC antibody assessment remains Uncertain (HPA: subcellular ICC-IF; HPA: antibody validation). | Use IF/ICC only as a localisation cross-check here. Interpret the paraffin-section chromogenic result using its own controls and record any compartment disagreement (HPA: subcellular ICC-IF; general IHC practice). |
Experiment evidence
IHC & IF images showing EFNA3 staining
A07595-1 · IHCImmunohistochemistry (IHC) analyzes of Ephrin-A3 (E196) pAb in paraffin-embedded human brain tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143590-EFNA3/tissue/cerebral+cortex#imid_3489344
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143590-EFNA3/tissue/colon#imid_3521446
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143590-EFNA3/tissue/liver#imid_3521447
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000143590-EFNA3/tissue/kidney#imid_3521448
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EFNA3 IHC & IF
🧪Run cerebral cortex first and look for staining in neuronal cells (HPA: High in neuronal cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); neighboring cells on the cortex slide that lack specific membrane staining can serve as internal negative cells without assigning them a presumed negative cell type (UniProt P52797: cell membrane).
Positive control tissue: Breast (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for EFNA3; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and a knockout sample or peptide-block control where available (standard IHC practice). Quench endogenous peroxidase for chromogenic IHC, and account for brain-tissue autofluorescence if assessing the cortex by IF (standard IHC/IF practice).
⚠️Feasibility: An EFNA3-specific fixation window and retrieval dependence are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections (standard IHC practice). The selected A07595-1 brain-tissue IHC caption reports paraffin embedding but leaves the fixative unreported (selected IHC caption). The evidence does not establish whether frozen sections or IF are easier; brain-tissue autofluorescence may complicate IF assessment (standard IF practice).
HPA tissue IHC evidence for EFNA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EFNA3 IHC Tips
Troubleshoot EFNA3 staining in paraffin sections by checking retrieval, signal location and tissue controls before interpreting chromogenic intensity.
Which retrieval conditions should I try first for EFNA3 in paraffin sections?
Start with citrate buffer at pH 6.0, heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Run a retrieval-free section alongside it to distinguish insufficient exposure from retrieval-associated background (standard IHC practice). If staining remains weak, titrate retrieval time on serial sections while keeping antibody concentration and detection conditions constant; record section loss and changes in background (standard IHC practice). Compare the resulting pattern with the expected cell-surface location of this GPI-anchored ligand, while allowing for the uncertain cytoplasmic pattern reported in tissue IHC (UniProt P52797 topology; HPA: reliability Uncertain).
Could fixation explain weak or uneven EFNA3 staining?
Target-specific fixation sensitivity is unknown: the selected antibody image identifies paraffin-embedded human brain tissue but does not state its fixative (A07595-1 tissue-IHC caption). Record the actual fixative, fixation duration and tissue-processing history for each section before comparing staining intensity (standard IHC practice). If material is available, compare matched sections processed under documented conditions and apply the same citrate pH 6.0, 95–98 °C, 20 min retrieval to each (page retrieval rule; standard IHC practice). Treat differences as processing-associated until independent controls establish that EFNA3 detection itself changed, and avoid inferring fixation effects from expression patterns or protein topology (standard IHC practice).
Where should convincing EFNA3 staining appear in tissue?
Prioritise staining at cell boundaries because EFNA3 is a cell-surface, GPI-anchored ligand with no transmembrane segment (UniProt P52797 topology and function). Its processed Ephrin-A3 chain spans residues 23–214, so the precursor description alone does not establish a nuclear or diffuse cytoplasmic staining pattern (UniProt P52797 processing). HPA reports cytoplasmic staining across several tissues, but assigns its tissue-IHC evidence uncertain reliability because staining and RNA data have low consistency and presumed off-target staining was observed (HPA: profile and reliability). Compare membrane-associated and cytoplasmic signals separately against controls before assigning either pattern to EFNA3 (standard IHC practice).
How do isoforms and epitope placement affect interpretation?
EFNA3 has 2 annotated isoforms, while the supplied product caption does not identify the antibody epitope or establish isoform selectivity (UniProt P52797 isoforms; A07595-1 tissue-IHC caption). The precursor has a signal peptide at residues 1–22, a processed chain at 23–214, and an Ephrin receptor-binding domain at 30–169 (UniProt P52797 processing and domains). Three annotated glycosylation sites occur at residues 38, 67 and 100 (UniProt P52797 glycosylation). Check the antibody’s documented immunogen or epitope before attributing differences between specimens to a particular isoform, processed region or glycosylation state (standard IHC practice).
How can IF help assess an ambiguous chromogenic EFNA3 pattern?
Use IF as a complementary localisation check alongside the paraffin-section IHC result, with a marker for the expected cell type; neuronal cells in cerebral cortex are one reported high-staining example, though HPA rates its tissue-IHC evidence uncertain (HPA: cerebral cortex neuronal cells High; HPA: reliability Uncertain). Choose a spectrally separated, preferably far-red fluorophore after checking tissue autofluorescence in an unstained control (standard IF practice). For an accessible extracellular epitope, compare staining without permeabilisation; for an intracellular epitope, titrate gentle permeabilisation, since the supplied caption does not locate this antibody’s epitope (UniProt P52797 topology; A07595-1 tissue-IHC caption; standard IF practice). Do not transfer the paraffin-image caption into an IF fixation claim (A07595-1 tissue-IHC caption).
What should I check when brown signal appears throughout the section?
First inspect no-primary and detection-only sections to locate staining from the chromogenic system rather than the primary antibody (standard IHC practice). For peroxidase-based detection, include an endogenous-peroxidase block before applying the DAB substrate and compare its effect on background (standard IHC practice). Titrate primary antibody concentration and blocking conditions on adjacent sections while keeping retrieval at citrate pH 6.0, 95–98 °C for 20 min (page retrieval rule; standard IHC practice). Be especially cautious about diffuse cytoplasmic signal: HPA describes that pattern but reports uncertain reliability and presumed off-target staining (HPA: profile and reliability).
How should I quantify EFNA3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then record the percentage of positive cells and staining intensity in each field (standard IHC practice). An H-score can combine intensity categories with their positive-cell percentages; report the scoring scale and use the same thresholds across specimens (standard IHC practice). For spatial questions, count positive cells per mm² of analyzable tissue and normalise to the relevant cell population or area, excluding folds and necrosis by preset rules (standard IHC practice). Report membrane-associated and cytoplasmic scores separately because UniProt places EFNA3 at the cell surface while HPA describes uncertain cytoplasmic tissue staining (UniProt P52797 topology; HPA: profile and reliability).
How can I distinguish genuine EFNA3 signal from artefact?
A cell-boundary pattern in an appropriate cell population is consistent with EFNA3’s GPI-anchored surface location, but morphology alone does not prove antibody specificity (UniProt P52797 topology; standard IHC practice). Check whether signal follows the expected cells rather than an unrelated compartment: HPA reports high neuronal-cell staining in cerebral cortex, while rating its overall tissue-IHC evidence uncertain (HPA: cerebral cortex neuronal cells High; HPA: reliability Uncertain). Exclude section-edge staining, necrotic regions and signal present in no-primary or peroxidase-control sections before scoring (standard IHC practice). Treat broad cytoplasmic staining cautiously because HPA reports that pattern alongside low staining–RNA consistency and presumed off-target binding (HPA: profile and reliability).
Boster reagents
Best EFNA3 / Ephrin-A3 IHC Antibodies
The catalog antibody has IHC image data from paraffin-embedded human brain tissue (catalog: A07595-1 IHC image caption). IF is listed, with Human, Mouse, and Rat reactivity (catalog: A07595-1 applications/reactivity).
Real IHC data
Anti-Ephrin-A3 (E196) EFNA3 Antibody
Cat # A07595-1
A07595-1 will render with an IHC image of paraffin-embedded human brain tissue (catalog: A07595-1 IHC image caption). Its listed applications include IHC and IF, and its listed reactivity covers Human, Mouse, and Rat (catalog: A07595-1 applications/reactivity).
Which to pick: For tissue IHC, choose A07595-1: its own image caption documents paraffin-embedded human brain tissue, but does not report the fixative (catalog: A07595-1 IHC image caption). For IF/ICC planning, A07595-1 lists IF at 1:50–1:200, but provides no IF image or ICC validation; its caption calls it a pAb, and no clone is specified (catalog: A07595-1 applications/IF dilution/IF image alts/IHC image caption/clone). For cross-species work, A07595-1 lists Human, Mouse, and Rat reactivity, while its IHC image documents only human tissue (catalog: A07595-1 reactivity/IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P52797 (EFNA3_HUMAN, Ephrin-A3).
- Human Protein Atlas. EFNA3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. EFNA3 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. EFNA3 antibody validation summary (1 antibodies).
- A molecular mechanism for the topographic alignment of convergent neural maps. eLife 2017 — PMC5360444.
- Clinicopathological and prognostic significance of Ephrin A3 in bladder urothelial carcinoma. Oncology letters 2023 — PMC10623084.
- EFNA3 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in patients with lung adenocarcinoma. Cancer cell international 2021 — PMC8513303.
- PRKN mediates the ubiquitination of SCAF8 to reduce the mRNA stability of KLF5 and its transcriptional activation of EFNA3 in colorectal cancer. Cellular & molecular biology letters 2026 — PMC13523511.
- PubMed PMID:7838529 — UniProt-cited evidence.
- PubMed PMID:7973638 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EFNA3 (Ephrin-A3). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A07595-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A07595-1); 2. Fixation: Image fixative and duration unreported (datasheet A07595-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-EFNA3, 1:50-1:200 (datasheet A07595-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining; reliability uncertain (HPA tissue IHC); Isoform / epitope: 2 isoforms; extracellular chain 23–214, no cytoplasmic tail (UniProt); Regulation: No specific regulatory cue reported (UniProt). Evidence: UniProt P52797, HPA tissue IHC · Uncertain, HPA ICC-IF · Membrane, Datasheet A07595-1, PMC10623084, PMC8513303. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EFNA3/immunohistochemistry-ihc-guide.